Haematopoietic Progenitor Cells
Haematopoietic progenitor cells (HPCs) are bone marrow derived stem cells that form the cellular constituents of blood (i.e. erythrocytes and leukocytes) and have the capacity to re-constitute bone marrow that has previously been ablated by irradiation. HPCs are used for bone marrow transplants (BMTs). HPCs are harvested from donors by mobilizing these cells into the blood. Currently the cytokine G-CSF is used to mobilize HPCs (Cashen et al, (2004) Curr. Haem. Rep. 3: 406-412) and it has been shown that maximal mobilization requires treatment daily over 4 consecutive days (or over 3-5 days), with blood being collected on day 5. However, in approximately 20% of patients not enough HPCs are mobilized to perform a BMT. Thus there is a need to find more effective reagents for mobilizing HPCs. The term Haematopoietic stem cell (HSC) is used interchangeably with HPC herein and references to HSC are intended to include reference to HPC.
CD34+ HPCs express the receptor CXCR4 and migrate in response to the chemokine stromal cell-derived factor (SDF)-1α (CXCL12) (Aiuti et al, (1997) J. Exp. Med. 185: 111-120). CXCR4 is the receptor for CXCL12, which is expressed constitutively in the bone marrow. There is evidence that CXCL12/CXCR4 is required for both the retention of HPCs within the bone marrow and their homing back to the bone marrow. AMD3100 is a specific CXCR4 antagonist and it stimulates a rapid rise in circulating numbers of HPCs in both mice and humans (Hatse et al, (2002) FEBS Letters 527: 255-262; Liles et al, (2003) Blood 102: 2728-2730; Broxmeyer et al, (2005) J. Exp. Med. 201: 1307-1318).
Recently it has been shown that the CXCR4 antagonist has the capacity to rapidly mobilize HPCs from the bone marrow within 1 h. Furthermore, we and others have shown a synergistic effect of chronic G-CSF treatment combined with acute treatment with AMD3100 with respect to HPC mobilization (Martin et al, (2006) Br. J. Haem. 134: 326-329; Broxmeyer et al, (2005) J. Exp. Med. 201: 1307-1318; Flomenberg et al, (2005) Blood 106: 1867-1874). Such a combined treatment is currently in phase III clinical trials for HPC mobilization.
Endothelial Progenitor Cells
Endothelial progenitor cells (EPCs) are bone marrow-derived progenitor cells that have been shown to contribute to vascularisation following tissue injury. In particular, following ischemia reperfusion injury such as occurs during myocardial infarction. EPCs contribute to the formation of new blood vessels and are thus thought to be clinically beneficial. The use of EPCs for treating conditions such as ischemic heart disease is reviewed in Seeger et al, (2007) Nature Clinical Practice: Cardiovascular Medicine 4: S110-S113.
An increase in EPCs in the blood is seen in patients with heart disease and correlates with the clinical outcome. It has been proposed that mobilizing EPCs from the bone marrow may be beneficial in such patients and a number of clinical trials have examined the effect of G-CSF treatment on mobilisation of stem cells in patients suffering acute myocardial infarction. Overall the results of these studies have been disappointing (Ince & Nienaber, (2007) Nature Clin. Pract 4 suppl 1: S114-S118; Takano H (2007) Trends Pharm. Sci. 28(10): 512-7 (Epub 2007 Sep. 20); and Ripa R S (2007) Circulation 116[suppl I]: I-24-I-30 and references cited therein). We believe that this may reflect the fact, as shown here, that G-CSF is not the most efficacious treatment to mobilize EPCs. Ripa et al. (2007) Circulation 116[suppl I]: I-24-I-30 shows that G-CSF mobilises HPCs but causes a decrease in MSCs. This may explain why G-CSF is not appropriate as a stem cell mobilising reagent. Furthermore G-CSF has other effects that may affect disease progression. Thus G-CSF has been shown to promote the survival of cardiac myocytes, which could also be beneficial in this disease setting. G-CSF also induces granulopoiesis and a dramatic rise in circulating neutrophils which may be detrimental to the heart, exacerbating the inflammatory process (Kang et al., (2004) Lancet. 363: 751-756). For these reasons it is difficult to interpret the previous and on-going trials using G-CSF to mobilize stem cells. Reagents that selectively mobilize EPCs from the bone marrow may be clinically useful in the treatment of heart disease (Ferrara et al., (2003) Nat. Medicine. 9: 669-676).
Mesenchymal Stem Cells
Mesenchymal stem cells (MSCs) are bone marrow-derived stem cells that have the capacity to differentiate into adipocytes, chondrocytes and osteocytes, ie exhibit tri-lineage differentiation. The tri-lineage differentiation of MSCs may be tested ex vivo as proof of the presence of MSCs. Some studies suggest that MSCs may be able to differentiate into other lineages, such as neuronal and epithelial cells. However, this is still controversial and not yet widely accepted. In the context of the present invention, it is expected that the MSCs exhibit said tri-linage differentiation under appropriate culture conditions, but they may also be able to differentiate into other cells.
We would also predict mobilised MSCs to retain the ability to differentiate into a more appropriate and therapeutically useful regenerative cell type depending on the tissue which was being targeted. It is possible that MSCs will differentiate into other lineages dependent on the disease/tissue environment, eg MSCs recruited into the damaged lung may differentiate into epithelial cells to repair damaged tissues. This is suggested in Ortiz et at (2003) PNAS USA 100(14): 8407-8411, which provides evidence that MSCs may be useful for tissue repair in respiratory diseases. Wang et at (2005) PNAS USA 102(1): 186-191 provides further evidence that MSCs possess the capacity of differentiating into airway epithelia, which suggests that MSC may provide a therapy for cystic fibrosis. As such it is believed that MSCs may be used clinically for tissue regeneration, for example, in the treatment of osteogenesis imperfecta. MSCs are reviewed in Roufosse et al, (2004) Intl. J. Biochem. & Cell Biol. 36: 585-597.
A recent review by Prockop D J ((2007) Clin. Pharmacol. Ther. 82(3): 241-3) encapsulates the current view that ability of MSCs to differentiate into different cell types (e.g. osteocytes or neurons) i.e. their “stemness”, may not be the reason why these cells are so effective in vivo at promoting tissue repair. The experimental evidence suggests that MSCs may repair tissues via their production of chemokines and cytokines. Potential mechanisms of tissue repair by MSCs are also discussed in Prockop at al (2003) PNAS USA 100 supp. 1: 11917-11923. Even though the mechanism of tissue repair has not yet been fully elucidated; it is clear that MSCs are therapeutically active and have many proven and potential uses.
EPCs and MSCs can be recognised in practice by a number of characteristics. Firstly, EPCs and MSCs only grow in defined culture media and colony growth takes on an easily identifiable morphology. Secondly, via FACS analysis, EPCs and MSCs can be further defined by the expression of distinct antigens. Antigen expression on MSCs is reviewed in Chamberlain et al., (2007) Stem cells. 25: 2739-2749; and Roufosse et al., (2004) International J. Biochem. & Cell. Biol. 36: 585-597. Antigen expression in EPCs is described in Yoon et al., (2005) Circulation. 112: 1618-1627; and Yoder et al., (2007) Blood 109: 1801-1809. A protocol for the detection, enumeration and phenotypic analysis of MSCs using flow cytometry is provided in Jones et al (2006) Cytometry Part B (Clinical Cytometry) 70B: 391-399.
Human MSCs are typically defined as cells that are:                1. Plastic adherent (ie adherent to typical plastic cell culture vessels);        2. Triple positive for the cell surface markers CD105, CD73 and CD90;        3. Negative for the cell surface markers, CD34, CD45, CD14, CD11b and CD19; and        4. Exhibit tri-lineage differentiation.        
Murine MSCs exhibit 1 and 4 (above), but the CD markers may be different (for references see above).
Human EPCs are typically defined as cells that are:                1. Adherent to tissue culture plasticware pre-coated with an extracellular matrix protein, e.g. fibronectin;        2. Positive for the cell surface markers CD34 or CD133 and VEGFR2; and        3. Uptake acetylated LDL and stain positive for GS-Lectin.        
Additionally it has been shown that MSCs are immunosuppressive. In vitro they have been shown to inhibit T cell proliferation, promote the differentiation of T cells into T reg cells and inhibit monocyte differentiation into dendritic cells. In vivo MSCs have been used in animal models for the treatment of diabetes, rheumatoid arthritis, systemic lupus erythematosus and multiple sclerosis. Finally MSCs have been shown to improve allogenic bone marrow engraftment following transplantation. It is believed that MSCs may be used for the treatment of autoimmune diseases, such as rheumatoid arthritis or sclerederma. Indeed to date >1000 people worldwide have received bone marrow transplants for autoimmune disease, for example for the treatment of therapy-resistant Graft Versus Host Disease or rejection of organ allographs. A review of the current and potential clinical uses of MSCs is provided in Giordano et al (2007) J. Cell. Physiol. 211: 27-35.
MSCs represent <0.001% of bone marrow cells (Tondreau et al, (2005) Stem Cells 23: 1105-1112). Isolation requires ex vivo expansion of MSCs in FCS containing growth medium. There are a number of problems associated with such isolation and expansion procedures, including the use of FCS, the potential of MSCs to transform and the possibility that such ex vivo culture may reduce the functionality of these cells. For example, see Procock D & Olson (2007) Blood. 109(8): 3147-51 (Epub 2006 Dec. 14), page 3150 indicates that MSCs expanded ex vivo may form tumours in vivo.
The factors regulating the mobilization of MSCs are currently unknown.
In the present application we identify factors regulating the mobilization of HPCs, EPCs and MSCs. We identify factors that selectively mobilize EPCs and/or MSCs are therefore useful for the clinical applications as described above.